Luke J. Mortensen

Glycoengineering of E‐Selectin Ligands by Intracellular versus Extracellular Fucosylation Differentially Affects Osteotropism of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (MSCs) hold great promise in cellular therapeutics for skeletal diseases but lack expression of E‐selectin ligands that direct homing of blood‐borne cells to bone marrow. Previously, we described a method to engineer E‐selectin ligands on the MSC surface by exofucosylating cells with fucosyltransferase VI (FTVI) and its donor sugar, GDP‐Fucose, enforcing transient surface expression of the potent E‐selectin ligand HCELL with resultant enhanced osteotropism of intravenously administered cells. Here, we sought to determine whether E‐selectin ligands created via FTVI‐exofucosylation are distinct in identity and function to those created by FTVI expressed intracellularly. To this end, we introduced synthetic modified mRNA encoding FTVI (FUT6‐modRNA) into human MSCs. FTVI‐exofucosylation (i.e., extracellular fucosylation) and FUT6‐modRNA transfection (i.e., intracellular fucosylation) produced similar peak increases in cell surface E‐selectin ligand levels, and shear‐based functional assays showed comparable increases in tethering/rolling on human endothelial cells expressing E‐selectin. However, biochemical analyses revealed that intracellular fucosylation induced expression of both intracellular and cell surface E‐selectin ligands and also induced a more sustained expression of E‐selectin ligands compared to extracellular fucosylation. Notably, live imaging studies to assess homing of human MSC to mouse calvarium revealed more osteotropism following intravenous administration of intracellularly‐fucosylated cells compared to extracellularly‐fucosylated cells. This study represents the first direct analysis of E‐selectin ligand expression programmed on human MSCs by FTVI‐mediated intracellular versus extracellular fucosylation. The observed differential biologic effects of FTVI activity in these two contexts may yield new strategies for improving the efficacy of human MSCs in clinical applications. Stem Cells 2016;34:2501–2511

Labeling and analysis of chicken taste buds using molecular markers in oral epithelial sheets

In chickens, the sensory organs for taste are the taste buds in the oral cavity, of which there are ~240–360 in total number as estimated by scanning electron microscopy (SEM). There is not an easy way to visualize all taste buds in chickens. Here, we report a highly efficient method for labeling chicken taste buds in oral epithelial sheets using the molecular markers Vimentin and α-Gustducin. Immediate tissue fixation following incubation with sub-epithelially injected proteases enabled us to peel off whole epithelial sheets, leaving the shape and integrity of the tissue intact. In the peeled epithelial sheets, taste buds labeled with antibodies against Vimentin and α-Gustducin were easily identified and counted under a light microscope and many more taste buds, patterned in rosette-like clusters, were found than previously reported with SEM. Broiler-type, female-line males have more taste buds than other groups and continue to increase the number of taste buds over stages after hatch. In addition to ovoid-shaped taste buds, big tube-shaped taste buds were observed in the chicken using 2-photon microscopy. Our protocol for labeling taste buds with molecular markers will factilitate future mechanistic studies on the development of chicken taste buds in association with their feeding behaviors.

Deep tissue single cell MSC ablation using a fiber laser source to evaluate therapeutic potential in osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a currently uncurable disease where a mutation in collagen type I yields brittle bones. One potential therapy is transplantation of mesenchymal stem cells (MSCs), but controlling and enhancing transplanted cell survival has proven challenging. Therefore, we use a 2- photon imaging system to study individual transplanted cells in the living bone marrow. We ablated cells deep in the bone marrow and observed minimal collateral damage to surrounding tissue. Future work will evaluate the local impact of transplanted MSCs on bone deposition in vivo.

Fast axial scanning for 2-photon microscopy using liquid lens technology

Scanning microscopy methods require movement of the focus in Z coordinates to produce an image of a 3-dimensional volume. In a typical imaging system, the optical setup is kept fixed and either the sample or the objective is translated with a mechanical stage driven by a stepper motor or a piezoelectric element. Mechanical Z scanning is precise, but its slow response and vulnerability to mechanical vibrations and stress make it disadvantageous to image dynamic, time-varying samples such as live cell structures. An alternative method less susceptible to these problems is to change the focal plane using conjugate optics. Deformable mirrors, acousto-optics, and electrically tunable lenses have been experimented with to achieve this goal and have attained very fast and precise Z-scanning without physically moving the sample. Here, we present the use of a liquid lens for fast axial scanning. Liquid lenses have a long functional life, high degree of phase shift, and low sensitivity to mechanical stress. They work on the principle of refraction at a liquid-liquid interface. At the boundary of a polar and an apolar liquid a spherical surface is formed whose curvature can be controlled by adjusting its relative wettability using electro-wetting. We characterize the effects of the lens on attainable Z displacement, beam spectral characteristics, and pulse duration as compared with mechanical scanning.

The Wave2 scaffold Hem-1 is required for transition of fetal liver hematopoiesis to bone marrow

The transition of hematopoiesis from the fetal liver (FL) to the bone marrow (BM) is incompletely characterized. We demonstrate that the Wiskott–Aldrich syndrome verprolin-homologous protein (WAVE) complex 2 is required for this transition, as complex degradation via deletion of its scaffold Hem-1 causes the premature exhaustion of neonatal BM hematopoietic stem cells (HSCs). This exhaustion of BM HSC is due to the failure of BM engraftment of Hem-1−/− FL HSCs, causing early death. The Hem-1−/− FL HSC engraftment defect is not due to the lack of the canonical function of the WAVE2 complex, the regulation of actin polymerization, because FL HSCs from Hem-1−/− mice exhibit no defects in chemotaxis, BM homing, or adhesion. Rather, the failure of Hem-1−/− FL HSC engraftment in the marrow is due to the loss of c-Abl survival signaling from degradation of the WAVE2 complex. However, c-Abl activity is dispensable for the engraftment of adult BM HSCs into the BM. These findings reveal a novel function of the WAVE2 complex and define a mechanism for FL HSC fitness in the embryonic BM niche.Hematopoietic stem cells (HSCs) migrate from the fetal liver to the bone marrow (BM) during embryogenesis. Here the authors show that the WAVE2 complex scaffold Hem1 is required for engraftment of HSCs in BM, not through its canonical role regulating actin polymerization, but through c-Abl survival signaling.

Resolution enhancement of 2-photon microscopy using high-refractive index microspheres

Intravital microscopy using multiphoton processes is the standard tool for deep tissue imaging inside of biological specimens. Usually, near-infrared and infrared light is used to excite the sample, which enables imaging several mean free path inside a scattering tissues. Using longer wavelengths, however, increases the width of the effective multiphoton Point Spread Function (PSF). Many features inside of cells and tissues are smaller than the diffraction limit, and therefore not possible to distinguish using a large PSF. Microscopy using high refractive index microspheres has shown promise to increase the numerical aperture of an imaging system and enhance the resolution. It has been shown that microspheres can image features ~λ/7 using single photon process fluorescence. In this work, we investigate resolution enhancement for Second Harmonic Generation (SHG) and 2-photon fluorescence microscopy. We used Barium Titanate glass microspheres with diameters ∼20–30 μm and refractive index ∼1.9–2.1. We show microsphere-assisted SHG imaging in bone collagen fibers. Since bone is a very dense tissue constructed of bundles of collagen fibers, it is nontrivial to image individual fibers. We placed microspheres on a dense area of the mouse cranial bone, and achieved imaging of individual fibers. We found that microsphere assisted SHG imaging resolves features of the bone fibers that are not readily visible in conventional SHG imaging. We extended this work to 2-photon microscopy of mitochondria in mouse soleus muscle, and with the help of microsphere resolving power, we were able to trace individual mitochondrion from their ensemble.

Characterization of bone collagen organization defects in murine hypophosphatasia using a Zernike model of optical aberrations

Bone growth and strength is severely impacted by Hypophosphatasia (HPP). It is a genetic disease that affects the mineralization of the bone. We hypothesize that it impacts overall organization, density, and porosity of collagen fibers. Lower density of fibers and higher porosity cause less absorption and scattering of light, and therefore a different regime of transport mean free path. To find a cure for this disease, a metric for the evaluation of bone is required. Here we present an evaluation method based on our Phase Accumulation Ray Tracing (PART) method. This method uses second harmonic generation (SHG) in bone collagen fiber to model bone indices of refraction, which is used to calculate phase retardation on the propagation path of light in bone. The calculated phase is then expanded using Zernike polynomials up to 15th order, to make a quantitative analysis of tissue anomalies. Because the Zernike modes are a complete set of orthogonal polynomials, we can compare low and high order modes in HPP, compare them with healthy wild type mice, to identify the differences between their geometry and structure. Larger coefficients of low order modes show more uniform fiber density and less porosity, whereas the opposite is shown with larger coefficients of higher order modes. Our analyses show significant difference between Zernike modes in different types of bone evidenced by Principal Components Analysis (PCA).

Two-photon deep-tissue spatially resolved mitochondrial imaging using membrane potential fluorescence fluctuations

Cell metabolism and viability are directly reflected in their mitochondria. Imaging-based analysis of mitochondrial morphological structure, size and dynamic characteristics can therefore provide critical insight into cell function. However, mitochondria are often very abundant, and due to their close to diffraction-limit size, it is often non-trivial to distinguish a tubular or large mitochondrion from an ensemble of punctate mitochondria. In this paper, we use membrane potential dependent fluorescence fluctuations of individual mitochondria to resolve them using an approach similar to single molecule localization microscopy. We use 2-photon microscopy to image mitochondrial intensity fluctuations at 200 μm deep inside an intact in-vivo mouse soleus muscle. By analyzing the acquired images, we can reconstruct images with an extra layer of information about individual mitochondria, separated from their ensemble. Our analysis shows a factor of 14 improvement in detection of mitochondria.

Finite difference time domain modeling of wavefront aberrations in bone using second harmonic generation microscopy (Conference Presentation)

Near infrared and infrared multi-photon imaging through or inside bone is an emerging field that promises to help answer many biological questions that require minimally invasive intravital imaging. Neuroscience researchers especially have begun to take advantage of long wavelength imaging to overcome multiple scattering and image deep inside the brain through intact or partially intact bone. Since the murine model is used in many biological experiments, here we investigate the optical aberrations caused by mouse cranial bone, and their effects on light propagation. We previously developed a ray tracing model that uses second harmonic generation in collagen fibers of bone to estimate the refractive index structure of the sample. This technique is able to rapidly provide initial information for a closed loop adaptive optics system. However, the ray tracing method does not account for refraction or scattering. Here, we extend our work to investigate the wavefront aberrations in bone using a full electromagnetic model. We used Finite-Difference Time-Domain modeling of light propagation in refractive index bone datasets acquired with second harmonic generation imaging. In this paper we show modeled wavefront phase from different originating points across the field of view.

Modelling of optical aberrations caused by light propagation in mouse cranial bone using second harmonic generation imaging

Multiphoton imaging through the bone to image into the bone marrow or the brain is an emerging need in the scientific community. Due to the highly scattering nature of bone, bone thinning or removal is typically required to enhance the resolution and signal intensity at the imaging plane. The optical aberrations and scattering in the bone significantly affect the resolution and signal to noise ratio of deep tissue microscopy. Multiphoton microscopy uses long wavelength (nearinfrared and infrared) excitation light to reduce the effects of scattering. However, it is still susceptible to optical aberrations and scattering since the light propagates through several layers of media with inhomogeneous indices of refraction. Mechanical removal of bone is highly invasive, laborious, and cannot be applied in experiments where imaging inside of the bone is desired. Adaptive optics technology can compensate for these optical aberrations and potentially restore the diffraction limited point spread function of the system even in deep tissue. To design an adaptive optics system, a priori knowledge of the sample structure assists selection of the proper correction element and sensing methods. In this work we present the characterization of optical aberrations caused by mouse cranial bone, using second harmonic generation imaging of bone collagen. We simulate light propagation through the bone, calculate aberrations and determine the correction that can be achieved using a deformable mirror.